Cytokinesis, the final event of the cell cycle, involves actomyosin based constriction of the plasma membrane to produce two daughter cells. Recent studies demonstrate that targeted membrane addition at the cleavage furrow also plays a key role in cytokinesis. This insight opens new avenues of investigation concerning the sources, sites, mechanisms and timing of membrane addition during cytokinesis. We address these issues by examining specialized and conventional cytokinesis furrows that form in Drosophila embryogenesis. A unique advantage of the early Drosophila embryo is that thousands of furrows synchronously form every fifteen minutes and conserved mechanisms driving membrane addition during cytokinesis can be analyzed through a combination of molecular genetic, cellular, and biochemical approaches. A major focus of this grant is to understand the mechanisms driving endocytic-based vesicle trafficking to the invaginating cytokinetic furrow. We also explore the mechanisms that link membrane addition to F-actin remodeling at the site of furrow formation and test the specific model that vesicles deliver potent actin remodelers to the site of furrow formation during the initial stages of cytokinesis. These issues will be addressed by examining interactions between Nuf, the homolog of the mammalian Rab11-effector FIP3, and Rab11 at the recycling endosome. Evidence suggests that cell cycle regulated Nuf:Rab11 interaction at the recycling endosome promotes both vesicle-mediated delivery and Rho-mediated actin polymerization at furrow. To test this, the dynamics of G and F-actin, activated Rho, and Rho-activators in normal and trafficking compromised embryos will be analyzed. Experiments are proposed to understand the role of cell cycle regulatory proteins that specifically influence Nuf recruitment to the recycling endosome as these will provide general insights into the cell cycle regulation of membrane addition at the cleavage furrow. Finally, we take advantage of a fortuitous convergence of exquisite cell biology, a comprehensive deficiency collection, and a shift from maternal to zygotic control during the first conventional cytokinesis in Drosophila embryogenesis to screen the genome for membrane trafficking components specifically required for cytokinesis. Identifying genes specifically required for membrane trafficking during cytokinesis will provides novel targets for designing drugs that selectively destroy rapidly dividing cancer cells.